Artificial auricle material with a composite vascularized and cartilaginous dual frame and preparation method thereof

Abstract

The present invention discloses an artificial auricle material with a dual framework of vascularization and chondrogenicity, and a preparation method thereof. The method comprises: 1) performing 3D modeling as needed to obtain an artificial auricle scaffold model with a large pore, and pre-setting a vascularization differentiation framework on the scaffold; 2) using multi-material multi-nozzle light-curing 3D printing technology, simultaneously printing the artificial auricle scaffold with a GelMA solution and printing a vascularization framework within the scaffold using a GelMA solution containing mesenchymal stem cells (MSCs); and culturing the vascularization differentiation to obtain an intermediate-form artificial auricle with the vascularization framework; 3) combining a multiple-immersion light-curing method, introducing a ColMA solution containing MSCs into the pores of the intermediate-form artificial auricle; and culturing the chondrogenic differentiation to obtain an artificial auricle material with both a vascularization and chondrogenicity framework. The simultaneous provision of vascularization and chondrogenicity frameworks within the scaffold provides a design paradigm for an artificial auricle with complete physiological functions.

Description

Technical Field

[0001] The present invention relates to the field of 3D photocuring printing and artificial auricle materials, and specifically to an artificial auricle material having a composite vascularized and cartilaginous dual framework and a preparation method thereof. Background Art

[0002] As a plastic and reconstructive medical device, artificial auricles are primarily used to replace lost or damaged auricle structures. Made from biocompatible materials, artificial auricles offer highly biomimetic visual effects, restoring the appearance of the patient's ear and boosting self-confidence. While they have achieved certain results in terms of aesthetics and functional restoration, the associated surgery and implants also face certain challenges, particularly infection risks and postoperative complications, which can place a physical and psychological burden on patients and increase the workload of physicians. Currently, there is still a lack of effective solutions to these issues.

[0003] In response to the above-mentioned issues, with the development of tissue engineering and materials science, chondrocyte-based artificial auricles are gradually moving from the laboratory to the clinic. This type of artificial auricle uses tissue engineering technology to culture, expand, and shape the patient's own or donated cartilage cells, ultimately creating an artificial auricle with a structure and function similar to the natural auricle. This technology not only restores the auricle's appearance but also improves biocompatibility, reducing the risk of rejection and postoperative infection. In addition, this type of artificial auricle is combined with biomaterials and 3D-printed scaffolds, and induced differentiation with a variety of cytokines to form an auricle scaffold that is more tailored to the individual patient and provides excellent conditions for chondrocyte attachment, growth, and differentiation. However, due to the thickness of the artificial auricle, the weak blood supply of the surrounding tissue cannot support the normal physiological function of the intermediate cells, resulting in hypoxic cell apoptosis and the formation of a necrotic core. This phenomenon accelerates cell apoptosis and disintegration of this type of artificial auricle, thereby hindering its long-term survival in the body. Therefore, in order to balance the strength of the cartilage structure and its ability to survive in the body, adding a microvascular structure that is conducive to the exchange of substances between cells into the cartilage structure is a necessary condition for the long-term survival of cartilage implants such as artificial auricles in the body.

[0004] Based on the above problems, the present invention combines a variety of advanced tissue engineering technologies. On the basis of the existing artificial auricle combined with 3D scaffolds and chondrocytes, it uses a preset structure for angiogenic differentiation and combines it with a 3D light-stereolithography-printed macroporous scaffold for chondrogenic differentiation, thereby obtaining an artificial auricle with a dual framework of vascularization and chondrogenesis. This thoroughly improves the blood supply and material exchange of the cartilage in the scaffold, and provides a new paradigm for the design of artificial auricles that can function in the body for a long time. In addition, the present invention can perform 3D reconstruction of the patient's auricle based on lidar and depth perception applications, providing a more user-friendly artificial auricle modeling solution. This solution greatly improves the efficiency of doctor-patient communication and can meet the patient's aesthetic and functional needs for ear structure reconstruction based on artificial auricle to a great extent during the design stage. Summary of the Invention

[0005] The purpose of the present invention is to provide an artificial auricle material with a dual framework of vascularization and cartilage and a preparation method thereof in view of the shortcomings of the existing technology and combining the advantages of various materials and processes.

[0006] The present invention is achieved by adopting the following technical solutions:

[0007] A method for preparing an artificial auricle material having a composite vascularized and cartilaginous dual framework comprises the following steps:

[0008] 1) Artificial auricle scaffold design: 3D modeling is performed as needed. For example, LiDAR combined with depth sensing can be used for 3D modeling. The patient's auricle is reconstructed to create an artificial auricle scaffold model with a pre-set vascular differentiation framework.

[0009] 2) Preparation of an intermediate form of an artificial auricle with a vascularized framework: Using multi-material, multi-nozzle, light-curing 3D printing technology, a GelMA solution is used to print the artificial auricle macroporous scaffold. A vascularized framework is then printed within the framework using a GelMA solution loaded with mesenchymal stem cells (MSCs). This is then cultured for angiogenic differentiation (usually for approximately 10 days) to obtain an intermediate form of the artificial auricle with a vascularized framework.

[0010] 3) Preparation of an artificial auricle based on a dual-frame: Combined with a multiple-immersion-photocuring method, a ColMA solution containing MSCs is introduced into the pores of an intermediate-form artificial auricle frame, and chondrogenic differentiation culture is performed (usually for about 4-6 weeks) to obtain an artificial auricle material with a dual-frame structure of vascularization and chondrogenesis.

[0011] Furthermore, in step 1), in a depth perception application used in a mobile device using a laser radar, the 3D modeling method includes one or more of a splattering method, a Gaussian mixture model, and a surface mesh subdivision method. The meshed artificial auricle macroporous scaffold model has a mesh scaffold diameter of 300-500 μm and a pore structure diameter of 200-300 μm. The pre-set angiogenic differentiation framework is a high-density angiogenic scaffold dispersed within the macroporous scaffold, with a scaffold diameter of 500-700 μm.

[0012] Furthermore, in step 2), the concentration of the GelMA solution for printing the artificial auricle scaffold is 15-20wt%, and the dissolution temperature is 60-80°C; the concentration of the GelMA solution for carrying mesenchymal stem cells is 5-7.5wt%, and the dissolution temperature is 60-80°C. After complete dissolution, it is cooled to 35-37°C; the photoinitiator added to the above two GelMA solutions is one or more of LAP, Irgacure, and I2959, with a concentration of 0.1-0.25wt%, and the dissolution is carried out under light-proof conditions. The MSCs are within the P2-P5 generation, with a concentration of 1×10 7 The concentration of 1000 / mL was dispersed in the low concentration GelMA solution.

[0013] Furthermore, in step 2), the multi-material multi-nozzle light-curing 3D printer uses a dual-nozzle dual-tank structure, the light source is a 350-410nm UV light source, and the light source power is 20-40mW / cm 2 The nozzle diameter for printing the auricle magnum scaffold is 300-500μm, the printing layer thickness is 300-500μm, and the layer interval is 200-300μm; the nozzle for printing the vascular differentiation framework is 500-700μm, and the printing layer thickness is 500-700μm; the printing temperature is 35℃-37℃, and the humidity is 60%-80%.

[0014] Furthermore, in step 2), the culture medium for angiogenic differentiation culture uses a basal culture medium of one or more of low-glucose DMEM or α-MEM, and the added angiogenic factors are one or more of VEGF, FGF-2, EGF and IGF-1, with a concentration range of 5-10 ng / ml.

[0015] Furthermore, in step 3), the ColMA solution has a concentration of 5-7.5 wt %, a dissolution temperature of 60-80° C., and is cooled to 35-37° C. after complete dissolution. The photoinitiator is one or more of LAP, Irgacure, and I2959, with a concentration of 0.1-0.25 wt %. The MSCs are of P2-P5 generations, and the concentration is 5×10 6 / ml; the basal culture medium for chondrogenic differentiation culture is one or more of low-glucose DMEM or α-MEM; the chondrogenic factors added to the ColMA solution and culture medium mainly include the following: one or more of TGF-β, BMPs, IGF-1 and FGF-2, with concentrations of 10-20ng / ml and 5-10ng / ml, respectively.

[0016] Furthermore, in step 3), the immersion time of the cyclic immersion-light curing method is 30-50s, and the light source during light curing is a UV light source with a wavelength of 350-410nm and a power of 20-40mW / cm 2 , the irradiation time is 10-15s, and the number of cycles is 3-5 times.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] 1) The present invention uses bioactive materials such as ColMA and GelMA as raw materials, loads mesenchymal stem cells, and combines a combination of multi-nozzle 3D light-curing printing, cyclic immersion-light-curing method and in vitro cartilage-induced differentiation to produce an artificial auricle structure with a dual framework of vascularization and cartilage. The raw material selection, process combination and system composition are innovative.

[0019] 2) The present invention uses lidar combined with depth perception applications to perform 3D modeling of the artificial auricle, replacing the traditional perfusion molding and CT-based modeling methods; compared with the perfusion molding modeling method, this type of 3D modeling method provides the possibility of customizing the artificial auricle shape. Compared with the CT modeling method, this method greatly simplifies the modeling cost and difficulty, and patients and doctors can customize the aesthetics and function of the artificial auricle through direct communication.

[0020] 3) The present invention is committed to preserving the mechanical structure of the chondrocyte artificial auricle while improving its long-term survival ability in the body. A triple gel structure is pre-designed during 3D modeling: a high-concentration GelMA macroporous framework provides a base structure for mesenchymal stem cells to attach and a major mechanical support scaffold structure; a second low-concentration GelMA framework is designed within the above-mentioned high-concentration GelMA macroporous framework. Its mild mechanical environment loads mesenchymal stem cells, which can greatly promote vascular differentiation and greatly improve the nutrient supply and waste exchange of the artificial auricle. The triple ColMA-loaded mesenchymal stem cells are loosely distributed in the pores of the macroporous scaffold through immersion-photocuring, and can form an artificial auricle with good biological activity and rigid structure through chondrogenic differentiation. The synergistic effect of the above-mentioned structures allows for a highly realistic simulation and reproduction of a natural auricle with vascular and cartilage structures, which has important clinical value and significance for promoting the development of artificial auricles.

[0021] 4) The artificial auricle material of the present invention, which incorporates a dual framework of vascularization and chondrogenesis, simultaneously achieves angiogenesis and chondrogenesis while maintaining the auricle's structural appearance and aesthetic appeal. Through an innovative step-by-step differentiation design, angiogenesis is completed within the framework, followed by chondrogenesis. The mild mechanical environment created by the low concentration of GelMA provides excellent conditions for the proliferation and differentiation of various cells, while the high concentration of GelMA forms a framework that not only provides excellent mechanical support but also a long degradation cycle, laying a solid foundation for the long-term function of the artificial auricle in the body.

[0022] 5) The present invention integrates a pre-defined vascular differentiation framework structure into a macroporous scaffold using a dual-nozzle, dual-tank 3D photopolymerization printing process. This process allows the vascular differentiation framework to be printed into the macroporous scaffold in a single step. The fibers formed by the vascular differentiation framework contain a large number of mesenchymal stem cells. During angiogenic differentiation, the mesenchymal stem cells migrate to the surface of the fibers, forming tubular structures as they differentiate. As differentiation progresses, the low-concentration GelMA in the scaffold degrades, ultimately forming a loose vascular structure that provides a stronger blood supply upon implantation.

[0023] 6) The 3D modeling process and the artificial auricle structure with a composite vascularized and cartilaginous dual frame in the present invention are applicable to almost all types of patients' technical requirements for artificial auricles and have strong universality. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the preparation process of the artificial auricle material with a composite vascularized and cartilaginous dual framework in the present invention;

[0025] Figure 2 Schematic diagram of the artificial auricle scan and reconstruction results. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments. Figure 1 The figure shows a schematic diagram of the process for preparing the artificial auricle material of the present invention.

[0027] Comparative Example 1:

[0028] 1) Artificial auricle support design: Figure 2 As shown, a 3D reconstruction of the patient's auricle was performed using the depth perception function of a 3D reconstruction device equipped with a LiDAR. This generated a 3D model file in OBJ format. This file was imported into Blender. Based on this model file, a meshed macroporous stent structure was designed. The stent diameter was approximately 300 μm, and the pore structure diameter was approximately 200 μm. This file was saved as an STL file for printing.

[0029] 2) Preparation of pre-printing solution: Under sterile, light-proof and heating conditions at 60°C, GelMA60 lyophilized powder was dissolved in a PBS solution containing 0.25 wt% LAP to prepare a 15 wt% high-concentration GelMA solution as a pre-printing solution for photocuring printing of the scaffold.

[0030] 3) Stereolithography Printing of the Artificial Ear Frame: Calibrate the 3D Stereolithography Printing Platform and load the STL model file. Load the pre-printing solution into the trough and use a high-concentration GelMA to print the artificial ear frame. The nozzle diameter for printing the artificial ear frame is 300 μm, the layer spacing is 200 μm, and the light source is a 410 nm UV light source with a light source power of 20 mW / cm. 2 , the printing temperature is 37℃ and the humidity is 60%.

[0031] 4) Artificial auricle framework into cartilage differentiation: Under sterile, dark-proof and 60°C conditions, ColMA solution was prepared by dissolving ColMA in PBS containing 0.25 wt% LAP, and P3 mesenchymal stem cells were cultured at a rate of 5×10 6 The density of cells / mL was dispersed in it, and then 20ng / ml TGF-β, BMPs, IGF-1 and FGF-2 were added to the ColMA solution. After the surface of the artificial auricle frame was dried, the solution was completely filled into the artificial auricle frame using the immersion-light curing method. After immersion for 30 seconds, a 410nm UV light source with a light source power of 20mW / cm 2 The artificial auricle material was obtained by irradiation for 10 seconds and repeated three times. The cells were then cultured in low-glucose DMEM medium containing 10 ng / ml TGF-β, BMPs, IGF-1 and FGF-2 for 6 weeks.

[0032] 5) Sections of the artificial auricle were sectioned and examined under a confocal microscope using live-dead staining, revealing numerous clusters of cells within the framework. 41% of these clusters showed a significant amount of red fluorescence, while 59% showed green fluorescence, suggesting the presence of a significant necrotic core. Toluidine blue staining of the sections to mark chondrocytes, followed by confocal fiber microscopy, revealed that no more than 15% of the cells were labeled, and these cells were distributed disorganized within the sections. These observations suggest that this type of artificial auricle material exhibits low cell survival and inefficient cartilage differentiation due to poor nutrient and waste exchange between the mesenchymal stem cells within the auricle.

[0033] Example 1:

[0034] 1) Artificial Auricle Scaffold Design: A 3D reconstruction of the patient's auricle was performed using the depth perception function of a 3D reconstruction device equipped with a LiDAR sensor. This generated a 3D model file in OBJ format. This file was imported into Blender software. Based on this model file, a gridded macroporous scaffold structure was designed with a scaffold diameter of approximately 300 μm and a pore structure diameter of approximately 200 μm. Within the macroporous scaffold, a high-density, dispersed vascularized scaffold with a diameter of approximately 500 μm was then designed. This file was saved as an STL file for printing.

[0035] 2) Preparation of printing pre-solution: GelMA60 lyophilized powder was dissolved in PBS solution containing 0.25 wt% LAP under sterile, light-proof and heating conditions at 60°C. 15 wt% high-concentration and 5 wt% low-concentration GelMA solutions were prepared respectively. P3 mesenchymal stem cells were cultured at a rate of 1×10 7 The concentration of cells / mL was dispersed in a low-concentration GelMA solution to obtain a low-concentration GelMA pre-solution with cells and a high-concentration GelMA pre-solution without cells.

[0036] 3) Preparation of an intermediate-form artificial auricle with a vascularized framework: Calibrate the 3D light-curing printing platform and load the STL model file. Load the low-concentration GelMA pre-solution containing cells and the high-concentration GelMA pre-solution without cells into the material tank. Use a dual-nozzle dual-tank structure installed in the light-curing 3D printer. The high-concentration GelMA solution prints the artificial auricle macroporous scaffold framework, and the low-concentration GelMA solution containing mesenchymal stem cells prints the preset vascularized scaffold (i.e., vascular framework); the nozzle diameter for printing the macroporous scaffold framework is 300μm, the layer spacing is 200μm, the nozzle diameter for printing the vascular framework is 500μm, the light source is a 410nm UV light source, and the light source power is 20mW / cm 2 The printing temperature was 37°C and the humidity was 60%. After printing, the entire scaffold was cultured in low-glucose DMEM medium containing 10ng / ml VEGF, FGF-2, EGF, and IGF-1 for 10 days. After angiogenic differentiation was completed, an intermediate-morphology artificial auricle with a vascularized framework was obtained.

[0037] 4) Preparation of artificial auricle based on double frame: ColMA solution was prepared by dissolving ColMA in PBS containing 0.25 wt% LAP under sterile, dark-proof and 60°C conditions, and mesenchymal stem cells at passage 3 were cultured at a rate of 5×10 6The density of cells / mL was dispersed in it, and then 20ng / ml TGF-β, BMPs, IGF-1 and FGF-2 were added to the ColMA solution; after the surface of the intermediate-shaped artificial auricle dried, the solution was completely filled into the entire auricle frame using the immersion-light curing method. After immersion for 30 seconds, a 410nm UV light source with a light source power of 20mW / cm 2 The cells were then irradiated for 10 seconds, repeated three times. The cells were then cultured in low-glucose DMEM medium containing 10 ng / ml TGF-β, BMPs, IGF-1, and FGF-2 for 6 weeks to obtain an artificial auricle material with a dual framework of vascularization and cartilage.

[0038] 5) Compared with Comparative Example 1, Example 1 presets a vascular framework in the artificial auricle large hole scaffold, and sequentially performs angiogenic and chondrogenic differentiation. The artificial auricle structure with a dual framework of vascularization and chondrogenesis was sliced, and live-dead staining was used under a confocal microscope to find that almost all cells were green fluorescent, accounting for as high as 89%, suggesting that the cells survive well in this type of substrate; the slices were stained with toluidine blue and von Willebrand factor (vWF) immunofluorescence staining to mark the chondrocytes and vascular endothelial cells of the artificial auricle with a dual framework of vascularization and chondrogenesis, respectively. Observation under a confocal fiberoscope revealed that the number of chondrocytes uniformly labeled with blue dye accounted for as high as 81%, while the vascular endothelial cells formed a uniform and continuous network structure, accounting for 8%. The above phenomenon suggests that the chondrocytes in the artificial auricle with a dual framework of vascularization and cartilage are evenly and densely differentiated, and present a good cartilage morphology; through the two-step differentiation method, vascular endothelial cells also form a dense vascular network in the framework. Such a structure lays the most basic histological structural foundation for the long-term survival of this type of artificial auricle in the body.

[0039] Example 2:

[0040] 1) Artificial auricle scaffold design: A 3D reconstruction of the patient's auricle was performed using the depth perception function of a 3D reconstruction device equipped with a LiDAR sensor. This generated a 3D model file in OBJ format. This file was imported into Blender software. Based on this model file, a gridded macroporous scaffold structure was designed with a scaffold diameter of approximately 300 μm and a pore structure diameter of approximately 500 μm. Within the macroporous scaffold, a high-density, dispersed vascularized scaffold with a diameter of approximately 500 μm was then designed. This file was saved as an STL file for printing.

[0041] 2) Preparation of printing pre-solution: GelMA60 lyophilized powder was dissolved in PBS solution containing 0.25 wt% LAP under sterile, light-proof and 60°C heating conditions. 15 wt% high concentration and 5 wt% low concentration GelMA solutions were prepared respectively. P3 mesenchymal stem cells were cultured at a density of 1×10 7 The concentration of cells / mL was dispersed in a low-concentration GelMA solution to obtain a low-concentration GelMA pre-solution with cells and a high-concentration GelMA pre-solution without cells.

[0042] 3) Preparation of the intermediate form of the artificial auricle with a vascularized framework: Calibrate the 3D light-curing printing platform and load the STL model file. Load the low-concentration GelMA pre-solution containing cells and the high-concentration GelMA pre-solution without cells into the material tank. Use a dual-nozzle dual-tank structure installed in the light-curing 3D printer. The high-concentration GelMA solution prints the artificial auricle macroporous scaffold framework, and the low-concentration GelMA solution containing mesenchymal stem cells prints the preset vascular framework. The nozzle diameter for printing the macroporous scaffold framework is 300μm, the layer spacing is 200μm, and the nozzle diameter for printing the vascular framework is 500μm. The light source is a 410nm UV light source with a light source power of 20mW / cm 2 The printing temperature was 37°C and the humidity was 60%. After printing, the entire scaffold was cultured in low-glucose DMEM medium containing 10ng / ml VEGF, FGF-2, EGF, and IGF-1 for 10 days. After angiogenic differentiation was completed, an intermediate-morphology artificial auricle with a vascularized framework was obtained.

[0043] 4) Preparation of artificial auricle based on double frame: ColMA solution was prepared by dissolving ColMA in PBS containing 0.25 wt% LAP under sterile, dark-proof and 60°C conditions, and mesenchymal stem cells at passage 3 were cultured at a rate of 5×10 6 The density of cells / mL was dispersed in it, and then 20ng / ml TGF-β, BMPs, IGF-1 and FGF-2 were added to the ColMA solution; after the surface of the intermediate-shaped artificial auricle dried, the solution was completely filled into the entire auricle frame using the immersion-light curing method. After immersion for 30 seconds, a 410nm UV light source with a light source power of 20mW / cm 2 The cells were then irradiated for 10 seconds, repeated three times. The cells were then cultured in low-glucose DMEM medium containing 10 ng / ml TGF-β, BMPs, IGF-1, and FGF-2 for 6 weeks to obtain an artificial auricle material with a dual framework of vascularization and cartilage.

[0044] 5) Compared with Example 1, Example 2 uses a pre-designed artificial auricle macroporous stent frame with a larger pore size. The artificial auricle structure with a dual vascularized and chondrogenic framework was sliced ​​and found under a confocal microscope using live-dead staining. The cells were almost all green fluorescent, accounting for as high as 94%, which suggests that the cells survive well in this type of substrate; the slices were stained with toluidine blue and von Willebrand factor (vWF) immunofluorescence staining to mark the chondrocytes and vascular endothelial cells of the artificial auricle with a dual vascularized and chondrogenic framework respectively. Observation under a confocal fiberscope revealed that the chondrocytes were uniformly labeled with blue dye, accounting for as high as 78%, while the vascular endothelial cells formed a uniform and continuous network structure, accounting for 6%. The above phenomenon suggests that the chondrocytes in the artificial auricle with a dual vascularized and chondrogenic framework are well differentiated, but a large number of holes are generated, and a dense cartilage morphology cannot be presented; through the two-step differentiation method, the vascular endothelial cells form a dense vascular network in the framework. Such a structure meets the histological structural basis for the long-term survival of the artificial auricle in the body. However, compared with Example 1, due to the excessively large pore size, the ColMA gel impregnated in the pore will degrade faster, causing the cells to detach from the auricle scaffold. The formed cartilage structure is too loose, which may lead to weak tissue mechanical properties.

[0045] Example 3:

[0046] 1) Artificial Auricle Scaffold Design: A 3D reconstruction of the patient's auricle was performed using the depth perception function of a 3D reconstruction device equipped with a LiDAR sensor. This generated a 3D model file in OBJ format. This file was imported into Blender software. Based on this model file, a gridded macroporous scaffold structure was designed with a scaffold diameter of approximately 300 μm and a pore structure diameter of approximately 200 μm. Within the macroporous scaffold, a high-density, dispersed vascularized scaffold with a diameter of approximately 500 μm was then designed. This file was saved as an STL file for printing.

[0047] 2) Preparation of printing pre-solution: GelMA60 lyophilized powder was dissolved in PBS solution containing 0.25 wt% LAP under sterile, light-proof and 60°C heating conditions. 15 wt% high concentration and 10 wt% low concentration GelMA solutions were prepared respectively. P3 mesenchymal stem cells were cultured at a density of 1×10 7 The concentration of cells / mL was dispersed in a low-concentration GelMA solution to obtain a low-concentration GelMA pre-solution with cells and a high-concentration GelMA pre-solution without cells.

[0048] 3) Preparation of the intermediate form of the artificial auricle with a vascularized framework: Calibrate the 3D light-curing printing platform and load the STL model file. Load the low-concentration GelMA pre-solution containing cells and the high-concentration GelMA pre-solution without cells into the material tank. Use a dual-nozzle dual-tank structure installed in the light-curing 3D printer. The high-concentration GelMA solution prints the artificial auricle macroporous scaffold framework, and the low-concentration GelMA solution containing mesenchymal stem cells prints the preset vascular framework. The nozzle diameter for printing the macroporous scaffold framework is 300μm, the layer spacing is 200μm, and the nozzle diameter for printing the vascular framework is 500μm. The light source is a 410nm UV light source with a light source power of 20mW / cm 2 The printing temperature was 37°C and the humidity was 60%. After printing, the entire scaffold was cultured in low-glucose DMEM medium containing 10ng / ml VEGF, FGF-2, EGF, and IGF-1 for 10 days. After angiogenic differentiation was completed, an intermediate-morphology artificial auricle with a vascularized framework was obtained.

[0049] 4) Preparation of artificial auricle based on double frame: ColMA solution was prepared by dissolving ColMA in PBS containing 0.25 wt% LAP under sterile, dark-proof and 60°C conditions, and mesenchymal stem cells at passage 3 were cultured at a rate of 5×10 6 The density of cells / mL was dispersed in it, and then 20ng / ml TGF-β, BMPs, IGF-1 and FGF-2 were added to the ColMA solution; after the surface of the intermediate-shaped artificial auricle dried, the solution was completely filled into the entire auricle frame using the immersion-light curing method. After immersion for 30 seconds, a 410nm UV light source with a light source power of 20mW / cm 2 The cells were then irradiated for 10 seconds, repeated three times. The cells were then cultured in low-glucose DMEM medium containing 10 ng / ml TGF-β, BMPs, IGF-1, and FGF-2 for 6 weeks to obtain an artificial auricle material with a dual framework of vascularization and cartilage.

[0050] 5) Compared to Example 1, Example 3 used a higher concentration of GelMA solution, 10 wt %, to print the vascular framework. Sections of the artificial auricle structure with a dual vascularized and chondrogenic framework were sliced ​​and examined under a confocal microscope using live-dead staining. The results showed that almost all cells exhibited green fluorescence, accounting for 78%, suggesting that the cells survived well in this type of substrate. Toluidine blue staining and von Willebrand factor (vWF) immunofluorescence staining were performed on the sections to mark the chondrocytes and vascular endothelial cells of the artificial auricle with a dual vascularized and chondrogenic framework, respectively. Confocal fiberoptic observation revealed that 74% of the chondrocytes were uniformly labeled with the blue dye, while the vascular endothelial cells formed a discontinuous network structure, accounting for 0.5%. The above phenomenon suggests that the chondrocytes in the artificial auricle with a dual framework of vascularization and chondrogenesis are evenly and densely differentiated and present a good cartilage morphology; however, due to the high concentration of the GelMA solution used in the preset vascular frame, the efficiency of angiogenic differentiation is low, and it is difficult to form a complete and continuous vascular network structure; compared with Example 1, this example may not provide a good exchange of nutrients and waste due to the discontinuous vascular network structure, which may affect its long-term survival in the body.

Claims

1. A method for preparing an artificial auricle material having a dual framework of vascularization and cartilage, characterized in that: The steps include: 1) Artificial auricle scaffold design: 3D modeling is performed as needed to obtain a gridded artificial auricle scaffold model with large holes, and a vascular differentiation framework is pre-set on it; 2) Preparation of an intermediate-morphology artificial auricle with a vascularized framework: Based on multi-material, multi-nozzle, light-curing 3D printing technology, a GelMA solution was used to print a large-pore artificial auricle scaffold according to the model in 1). At the same time, a GelMA solution containing mesenchymal stem cells (MSCs) was used to print a vascularization differentiation framework within the framework. After vascularization differentiation culture, an intermediate-morphology artificial auricle with a vascularized framework was obtained. 3) Preparation of an artificial auricle based on a dual framework: Combined with a multiple immersion-photocuring method, a ColMA solution containing MSCs was introduced into the pores of an intermediate-form artificial auricle. After chondrogenic differentiation culture, an artificial auricle material with both a vascularized and chondrogenic dual framework was obtained.

2. The method for preparing an artificial auricle material having a composite vascularized and cartilaginous dual framework according to claim 1, characterized in that: In step 1), the mesh scaffold in the meshed artificial auricle macroporous scaffold model has a diameter of 300-500 μm and a pore structure diameter of 200-300 μm; the preset angiogenic differentiation framework is angiogenic scaffolds dispersed in the macroporous scaffold, and the scaffold diameter is 500-700 μm.

3. The method for preparing an artificial auricle material having a composite vascularized and cartilaginous dual framework according to claim 1, characterized in that: In step 2), the concentration of the GelMA solution for printing the artificial auricle scaffold is 15-20wt%, and the dissolution temperature is 60-80°C; the concentration of the GelMA solution for carrying mesenchymal stem cells is 5-7.5wt%, and the dissolution temperature is 60-80°C. After complete dissolution, it is cooled to 35-37°C; a photoinitiator is added to the above two GelMA solutions, which is one or more of LAP, Irgacure, and I2959, with a concentration of 0.1-0.25wt%, and the dissolution is carried out under dark conditions; the MSCs are within the P2-P5 generation, and the concentration is 1×10 7 The concentration of 1000 μg / mL was dispersed in the GelMA solution.

4. The method for preparing an artificial auricle material having a composite vascularized and cartilaginous dual framework according to claim 1, characterized in that: In step 2), the multi-material multi-nozzle light-curing 3D printer uses a dual-nozzle dual-tank structure, the light source is a 350-410nm UV light source, and the light source power is 20-40mW / cm 2 , the nozzle diameter for printing the artificial auricle magnum scaffold is 300-500μm, the printing layer thickness is 300-500μm, and the layer interval is 200-300μm; The nozzle for printing the angiogenic differentiation framework is 500-700 μm, the printing layer thickness is 500-700 μm; the printing temperature is 35°C-37°C, and the humidity is 60%-80%.

5. The method for preparing an artificial auricle material having a composite vascularized and cartilaginous dual framework according to claim 1, characterized in that: In step 2), the medium for the angiogenic differentiation culture uses a basal medium of one or more of low-glucose DMEM or α-MEM, and the added angiogenic factors are one or more of VEGF, FGF-2, EGF and IGF-1, with a concentration range of 5-10 ng / ml.

6. The method for preparing an artificial auricle material having a composite vascularized and cartilaginous dual framework according to claim 1, characterized in that: In step 3), the ColMA solution has a concentration of 5-7.5 wt %, a dissolution temperature of 60-80°C, and is cooled to 35-37°C after complete dissolution. The photoinitiator added is one or more of LAP, Irgacure, and I2959, with a concentration of 0.1-0.25 wt %. The MSCs are of P2-P5 generations, and the concentration is 5×10 6 pcs / ml.

7. The method for preparing an artificial auricle material having a composite vascularized and cartilaginous dual framework according to claim 1, characterized in that: In step 3), the basal medium for chondrogenic differentiation culture is one or more of low-glucose DMEM or α-MEM; the chondrogenic factors added to the ColMA solution and the culture medium mainly include the following: one or more of TGF-β, BMPs, IGF-1 and FGF-2, the concentration of the chondrogenic factors in the ColMA solution is 10-20 ng / ml, and the concentration of the chondrogenic factors in the culture medium is 5-10 ng / ml.

8. The method for preparing an artificial auricle material having a composite vascularized and cartilaginous dual framework according to claim 1, characterized in that: In step 3), the immersion time of the cyclic immersion-light curing method is 30-50s, and the light source during light curing is a UV light source with a wavelength of 350-410nm and a power of 20-40mW / cm 2 , the irradiation time is 10-15s, and the number of cycles is 3-5 times.

9. An artificial auricle material with a composite vascularized and cartilaginous dual framework, characterized in that: The method is prepared by the method according to any one of claims 1 to 8.

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